DOK7 Mutations Research Articles

Congenital Myasthenic Syndrome: case study from a tertiary care institute of Western India

Objective. To describe clinical characteristics, genetic details and management of patients of congenital myasthenic syndrome. Methods. A retrospective study was carried out from 2020 to 2023. Patient who presented with ptosis, ophthalmoplegia, proximal limb weakness with genetically confirmed mutations for CMS were included in the study. Patients who were positive for Acetylcholine receptor antibody (AChRAb) or Muscle specific kinase (MuSK) antibody or any other antibodies suggestive of acquired myasthenia were excluded from the study. Outcome. 6 genetically proven patients were identified out of which 4 patients had CHRNE mutation, 1 patient had CHRNA1 mutation and 1 patient had Dok7 mutation. Mean delay in diagnosis was 15 years. Patient with Dok 7 mutation responded to salbutamol, 1 patient of CHRNE mutation required combination of pyridostigmine, fluoxetine and salbutamol, rest all patient responded to pyridostigmine. It took 4 weeks for improvement after starting salbutamol in patient with Dok7 mutation. Conclusion. Although rare, CMS is potentially treatable group of disease. Patient presenting with early onset muscular weakness should be evaluated thoroughly and CMS should be considered in presence of eyelid ptosis, ophthalmoplegia and proximal weakness with diurnal fluctuations. RNS and genetic testing are paramount in achieving an early diagnosis.

A novel DOK7 mutation causing limb-girdle congenital myasthenic syndrome

We report a case series of 5 Latino patients with limb-girdle pattern weakness, four patients are sisters, with one patient unrelated. Repetitive nerve stimulation showed a significant decrement in all cases. Targeted genetic testing for congenital myasthenic syndromes demonstrated a known DOK7 pathogenic mutation in each case, and in all five cases also revealed a novel DOK7 missense mutation in exon 7 with c.94G>A; providing strong evidence this mutation is pathogenic. DOK7-related congenital myasthenic syndrome often lacks oculobulbar involvement, and may present with limb-girdle weakness, mimicking limb-girdle muscular dystrophy.

Mechanism of disease and therapeutic rescue of Dok7 congenital myasthenia

Congenital myasthenia (CM) is a devastating neuromuscular disease, and mutations in DOK7, an adaptor protein that is crucial for forming and maintaining neuromuscular synapses, are a major cause of CM1,2. The most common disease-causing mutation (DOK71124_1127 dup) truncates DOK7 and leads to the loss of two tyrosine residues that are phosphorylated and recruit CRK proteins, which are important for anchoring acetylcholine receptors at synapses. Here we describe a mouse model of this common form of CM (Dok7CM mice) and a mouse with point mutations in the two tyrosine residues (Dok72YF). We show that Dok7CM mice had severe deficits in neuromuscular synapse formation that caused neonatal lethality. Unexpectedly, these deficits were due to a severe deficiency in phosphorylation and activation of muscle-specific kinase (MUSK) rather than a deficiency in DOK7 tyrosine phosphorylation. We developed agonist antibodies against MUSK and show that these antibodies restored neuromuscular synapse formation and prevented neonatal lethality and late-onset disease in Dok7CM mice. These findings identify an unexpected cause for disease and a potential therapy for both DOK7 CM and other forms of CM caused by mutations in AGRIN, LRP4 or MUSK, and illustrate the potential of targeted therapy to rescue congenital lethality.

Congenital myasthenic syndrome due to DOK7 mutation in a cohort of patients with ‘unexplained’ limb-girdle muscular weakness

Congenital myasthenic syndromes (CMS) associated with pathogenic variants in the DOK7 gene (DOK7-CMS) have phenotypic overlap with other neuromuscular disorders associated with limb-girdle muscular weakness (LGMW). Genetic analysis of the most common mutation (c.1124_1127dupTGCC) in DOK7 was performed in 34 patients with “unexplained” LGMW associated with non-specific changes in muscle biopsy. Of the 34 patients, one patient showed the DOK7 c.1124_1127dupTGCC variant in homozygousity. Our study estimates the minimum prevalence of undiagnosed DOK7-CMS to be 2.9% in southern Brazilian patients from our centre. Our data confirm that clinicians should look for DOK7-CMS patients when the clinical manifestation is an ‘unexplained’ LGMW, mainly if associated with non-specific changes in muscle biopsy.

Intrafamilial variation in clinical manifestations and response to salbutamol in siblings with congenital myasthenic syndrome caused by DOK7 mutations

The congenital myasthenic syndromes (CMS) are a heterogeneous group of disorders arising from genetic defects in presynaptic, synaptic, and postsynaptic proteins of the neuromuscular junction (NMJ) resulting in variable and characteristically fatigable muscle weakness affecting limb, ocular, bulbar, trunk, and respiratory muscles from early life. DOK7 mutation resulting in synaptic and postsynaptic CMS clinically presents with limb-girdle myasthenia with sparing of facial and EOM. They characteristically worsen with conventional treatment and show excellent response to salbutamol/ ephedrine. Here we present a case highlighting a varied presentation beginning in late childhood and its evolution to reveal its congenital nature and subsequent management with salbutamol.

Trouble at the junction: When myopathy and myasthenia overlap.

Although myopathies and neuromuscular junction disorders are typically distinct, their coexistence has been reported in several inherited and acquired conditions. Affected individuals have variable clinical phenotypes but typically display both a decrement on repetitive nerve stimulation and myopathic findings on muscle biopsy. Inherited causes include myopathies related to mutations in BIN1, DES, DNM2, GMPPB, MTM1, or PLEC and congenital myasthenic syndromes due to mutations in ALG2, ALG14, COL13A1, DOK7, DPAGT1, or GFPT1. Additionally, a decrement due to muscle fiber inexcitability is observed in certain myotonic disorders. The identification of a defect of neuromuscular transmission in an inherited myopathy may assist in establishing a molecular diagnosis and in selecting patients who would benefit from pharmacological correction of this defect. Acquired cases meanwhile stem from the co-occurrence of myasthenia gravis or Lambert-Eaton myasthenic syndrome with an immune-mediated myopathy, which may be due to paraneoplastic disorders or exposure to immune checkpoint inhibitors.

CONGENITAL MYASTHENIC SYNDROMES AND MYASTHENIA: P.73Characterization of an Indian congenital myasthenic syndrome cohort by whole exome sequencing

Congenital myasthenic syndromes (CMS) are caused by mutations in genes coding for proteins involved in the maintenance of the neuromuscular junction (NMJ). The patients can be effectively treated and prognosis can be improved when the precise genetic cause is known. In addition, the frequency of the different CMS-causing genes varies with ethnic and geographic origin. There are only few reports from India describing the occurrence of DOK7, RAPSN and COLQ mutations based on selective or limited genetic testing, but the prevalence of various gene mutations in the Indian CMS population is unknown, hence, limiting the effective gene screening and thus, prognosis and access to treatments. DNA from 40 genetically undiagnosed CMS families (total of 87 samples) were subjected to whole exome sequencing (WES). Of those eight families were known to be consanguineous, and majority of them had recessive inheritance pattern.WES was performed using Illumina exome capture (38 Mb target). Data analysis was carried out on the Seqr Genome Analysis Platform. Standard filtering criteria were moderate to high VEP (variant effect predictor), MAF of 0.01(1%) and CADD >20.We identified four GFPT1, four DPAGT1, three COLQ, two MUSK, one CHRND and one SLC25A1 variants that we consider as likely disease-causing. All the variants were highly pathogenic and majority of variants were absent in the control population. The overall solved rate after initial screening for known CMS genes was 37.5% cases (15 out of 40). Interestingly, there was occurrence of rare forms of CMS in the present cohort, and several recurrent mutations seen in COLQ, MUSK and DPAGT1 genes. These mutations might be hot-spots or founder mutations in Indian populations. The remaining patients will undergo further analysis for novel candidate genes.

P.415 - Congenital myasthenia syndromes: clinical description of a pediatric cohort

Congenital myasthenic syndromes (CMS) are a group of heterogeneous diseases caused by mutations in genes encoding for proteins involved in the neuromuscular junction that lead to an impaired signal transmission. We describe the clinical and paraclinical history of 12 patients with genetically confirmed CMS (1 CHRND, 2 AGRN, 1 DOK7, 1 MUSK, 4 RAPSN, 1 DPAGT1, 2 COLQ). Fatigable weakness was mentioned in all patients. The first symptom appeared at the latest in the first month of life (extremes: antenatal to less than 1 month of life). 4 patients had neonatal stridor. 11 patients had at least one severe respiratory decompensation with intubation. 8 had been tracheotomized. One patient died (COLQ mutation). 5 patients had arthrogryposis, 1 postural syndrome. Ptosis was reported in 11 patients. 9 of 10 patients who had reached the age of gait walked, (average age: 40 months; median age: 24 months). All patients with RAPSN mutation had clinodactyly. Electromyographic analysis showed neuromuscular junction abnormalities in 9 of 11 patients. Stimulated single fiber was always abnormal (done in 5 patients). Muscle biopsy exhibited mainly type 1 fibers predominance. Histology study of motor endplate performed in 2 patients was abnormal. Diagnostic delay was 35 months on average with a median at 9 months. From a therapeutic point of view, patients with mutations in the RAPSN gene were all significantly improved by anticholinesterasic monotherapy. A bi or triple therapy was required in the others (anticholinesterase, salbutamol / ephedrine, 3–4 DAP). Anticholinesterase drugs aggravated myasthenic syndrome in 3 patients (AGRN, DOK7 and COLQ mutations). The diagnosis of SMC is challenging due to life threatening complications, especially since a specific treatment exists.

Recent advances in congenital myasthenic syndromes

AbstractCongenital myasthenic syndromes (CMS) are heterogeneous disorders caused by germline mutations in genes expressed at the neuromuscular junction. Mutations have been identified in 24 genes encoding acetylcholine receptor subunits (CHRNA1, CHRNB1, CHRND, CHRNE and CHRNG), skeletal muscle sodium channel (SCN4A), signaling molecules driving acetylcholine receptor subunits clustering (AGRN, LRP4, MUSK and DOK7), synaptic structural proteins (COLQ, LAMB2 and COL13A1), postsynaptic structural proteins (RAPSN and PLEC), presynaptic molecules (CHAT, SYT2), glycosylation enzymes (GFPT1, DPAGT1, ALG2, ALG14 and GMPPB), and other less characterized molecules (PREPL and SCL25A1). CMS are recessive disorders, except for slow channel CMS and synaptotagmin 2 (SYT2)‐CMS. Onsets are largely less than 2 years, but adult‐onset is not rare, especially in slow‐channel CMS and limb‐girdle type CMS caused by glycosylation defects and by DOK7 mutations. Clinical features include fatigable muscle weakness, amyotrophy and minor facial anomalies. Eye, facial and bulbar muscles are frequently affected, but sparing of these muscles is observed, especially in limb‐girdle type CMS. Serum creatine kinase levels are frequently elevated in slow‐channel CMS, glutamine‐fructose‐6‐phosphate aminotransferase 1 (GFPT1)‐CMS, and guanosine diphosphate mannose pyrophosphorylase B (GMPPB)‐CMS. Electrophysiological findings supporting compromised neuromuscular signal transmission are a prerequisite for diagnosing CMS. Most CMS patients are likely to be underdiagnosed, and recognition of CMS in undiagnosed muscle weakness and/or amyotrophy is critical for diagnosing CMS.

Anticholinesterase Therapy Worsening Head Drop and Limb Weakness Due to a Novel DOK7 Mutation

Dok-7 myasthenia is an autosomal recessive congenital myasthenic syndrome due to DOK7 mutations. Anticholinesterase therapy is ineffective and may worsen the weakness in patients with Dok-7 myasthenia or few other forms of congenital myasthenic syndromes. We describe a 31-year-old man previously diagnosed with seronegative myasthenia gravis. Repetitive stimulation of the right spinal accessory nerve showed 51% decrement. Needle electromyography revealed myopathic changes in clinically affected muscles. Muscle biopsy was normal. The patient was referred to us for worsening weakness after taking pyridostigmine. We searched for DOK7 mutations and identified compound heterozygous mutations of a common c.1124_1127dupTGCC mutation and a novel splice site mutation, c.772+2_+4delinsCCGGGCAGGCGGGCA. Discontinuation of pyridostigmine improved weakness. He further regained strength with oral albuterol therapy and decrement was reduced to 25%. Worsening of symptoms with anticholinesterase therapy in patients with "seronegative myasthenia gravis" should prompt clinicians to consider a possibility of congenital myasthenic syndromes to avoid unnecessary use of immunosuppressive therapy. Patients with Dok-7 myasthenia respond well to oral albuterol treatment.

Global N-linked Glycosylation is Not Significantly Impaired in Myoblasts in Congenital Myasthenic Syndromes Caused by Defective Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1).

Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is an essential substrate for all mammalian glycosylation biosynthetic pathways and N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. It has been reported that GFPT1 mutations lead to a distinct sub-class of congenital myasthenic syndromes (CMS) termed “limb-girdle CMS with tubular aggregates”. CMS are hereditary neuromuscular transmission disorders in which neuromuscular junctions are impaired. To investigate whether alterations in protein glycosylation at the neuromuscular junction might be involved in this impairment, we have employed mass spectrometric strategies to study the N-glycomes of myoblasts and myotubes derived from two healthy controls, three GFPT1 patients, and four patients with other muscular diseases, namely CMS caused by mutations in DOK7, myopathy caused by mutations in MTND5, limb girdle muscular dystrophy type 2A (LGMD2A), and Pompe disease. A comparison of the relative abundances of bi-, tri-, and tetra-antennary N-glycans in each of the cell preparations revealed that all samples exhibited broadly similar levels of branching. Moreover, although some differences were observed in the relative abundances of some of the N-glycan constituents, these variations were modest and were not confined to the GFPT1 samples. Therefore, GFPT1 mutations in CMS patients do not appear to compromise global N-glycosylation in muscle cells.

Congenital Myasthenic Syndromes with Predominant Limb Girdle Weakness.

Congenital myasthenic syndromes are a heterogeneous group of genetically determined disorders characterized by impaired neuromuscular transmission. They usually present from birth to childhood and are characterised by exercise induced weakness and fatigability. Genotype-phenotype correlations are difficult. However, in some patients particular phenotypic aspects may point towards a specific genetic defect.The absence of ptosis and ophthalmoparesis in patients with limb-girdle weakness makes the diagnosis of a neuromuscular transmission defect particularly challenging (LG-CMS). This is illustrated by a well-documented case published by Walton in 1956. The diagnosis of LG-CMS is secured by demonstrating a neuromuscular transmission defect with single fibre EMG or repetitive nerve stimulation, in the absence of auto-antibodies. Ultimately, a genetic test is required to identify the underlying cause and assure counselling and optimization of treatment. LG-CMS are inherited in autosomal recessive traits, and are often associated with mutations in DOK7 and GFPT1, and less frequently with mutations in COLQ, ALG2, ALG14 and DPAGT.Genetic characterization of CMS is of the upmost importance when choosing the adequate treatment. Some of the currently used drugs can either ameliorate or aggravate the symptoms depending on the underlying genetic defect. The drug most frequently used for the treatment of CMS is pyridostigmine an acetylcholinesterase inhibitor. However, pyridostigmine is not effective or is even detrimental in DOK7- and COLQ-related LG-CMS, while beta-adrenergic agonists (ephedrine, salbutamol) show some sustained benefit. Standard clinical trials may be difficult, but standardized follow-up of patients and international collaboration may help to improve the standards of care of these conditions.

PP09.8 – 2377: Novel DOK7 mutation in a Greek patient with congenital myasthenic syndrome

Introduction Congenital myasthenic syndromes (CMS) are rare genetic syndromes resulting from presynaptic, synaptic or postsynaptic defects of the neuromuscular junction. In 2006 mutations in DOK7, a gene encoding a muscle-intrinsic activator of MUSK, were proven a postsynaptic cause of CMS. Until today 65 DOK7 mutations have been described. Case report A 16 year old girl, daughter to healthy, non-consanguineous parents has been followed up at our clinic since birth. History includes decreased fetal movements, hypotonia at birth and slightly delayed motor milestones with sitting at 8 months and walking at 15 months. Neurological examination discloses facial and proximal muscle weakness, bulbar and respiratory weakness, ptosis, joint laxity, massive kyphoscoliosis, feeding difficulty and failure to thrive. The patient is currently ambulant, able to climb stairs and walk approximately 50 meters, before getting tired. Under nocturnal noninvasive ventilation (BiPAP) she maintains oxygen saturation levels >98%. Creatin kinase is 200 U/l (normal: 150 U/l). Electrophysiologic studies revealed normal NCV's and a mildly myopathic EMG. Repetitive nerve stimulation demonstrated mild decrement at infancy, but now is normal. Muscle biopsy showed predominance of type 1 fibers and decreased oxidative activity. Since the clinical features were consistent with CMS, molecular analysis of CHAT, RAPSN, CHRNE and DOK7 was undertaken, which in turn revealed a compound heterozygous mutation in the DOK7 gene. Conclusion To the best of our knowledge this is the first Greek patient with confirmed DOK7 myasthenic syndrome. One of the mutations, the c.472C>T (p.Arg158Trp) has not been previously described. Accurate diagnosis of DOK7 myasthenic syndromes bears important clinical implications, since ephedrine or salbutamol rather than acetylcholinesterase inhibitors are the treatment of choice.

Congenital Myasthenic Syndrome due to DOK7 Mutations in a Family from Chile (P2.014)

Congenital Myasthenic Syndrome due to DOK7 Mutations in a Family from Chile (P2.014)

Limb girdle weakness responding to salbutamol: an Indian family with DOK7 mutation.

Congenital Myasthenic Syndromes (CMS) are heterogeneous genetic diseases. Two siblings presented with progressive limb girdle weakness without significant fluctuations or ocular muscle weakness. Repetitive nerve stimulation showed a decremental response and there was no response to pyridostigmine therapy. A trial of salbutamol produced a remarkable, consistent improvement. Mutation in exon 5 of the DOK7 gene was found in both siblings. Patients with congenital myasthenic syndrome with DOK 7 mutation benefit remarkably with salbutamol.

Interactions of Dok7 with Model Membranes Containing Anionic Lipids and Phosophoinositides

The receptor tyrosine kinases (RTKs) are a major class of transmembrane receptors responsible for regulation of many biological processes, including development and maintenance of synapses. Many RTKs act in concert with intracellular proteins which are thought to interact with cell membranes. Downstream-of-Kinase 7 (Dok7) is a soluble protein involved in the Muscle-Specific Kinase (MuSK) signalling pathway. Dok7 binds to MuSK to facilitate clustering of acetylcholine receptors at synapses. Mutations in Dok7 have been known to result in varying degrees of severity of congential myasthenic syndromes, which is characterised by impaired muscle contractions. The structure of Dok7 reveals it to contain a pleckstrin-homology (PH) and a phosphotyrosine-binding (PTB) domain. Although there is evidence to suggest how Dok7 interacts with MuSK, it is unknown exactly how it interacts with the cell membrane.Here we apply a multi-scale Molecular Dynamics simulations method to characterise Dok7's interactions with membranes of varying lipid composition. Coarse-grained (CG) simulations are used to characterise the mechanism of which Dok7 binds to the bilayer, while atomistic simulations are used to refine its interactions with specific lipids present in the bilayer. These computational studies reveal the role of phosphatidylinositol phosphates (PIP) in the interaction of Dok7 with complex cell membranes.

Identification of a Dutch founder mutation in MUSK causing fetal akinesia deformation sequence.

Fetal akinesia deformation sequence (FADS) refers to a clinically and genetically heterogeneous group of disorders with congenital malformations related to impaired fetal movement. FADS can result from mutations in CHRNG, CHRNA1, CHRND, DOK7 and RAPSN; however, these genes only account for a minority of cases. Here we identify MUSK as a novel cause of lethal FADS. Fourteen affected fetuses from a Dutch genetic isolate were traced back to common ancestors 11 generations ago. Homozygosity mapping in two fetuses revealed MUSK as a candidate gene. All tested cases carried an identical homozygous variant c.1724T>C; p.(Ile575Thr) in the intracellular domain of MUSK. The carrier frequency in the genetic isolate was 8%, exclusively found in heterozygous carriers. Consistent with the established role of MUSK as a tyrosine kinase that orchestrates neuromuscular synaptogenesis, the fetal myopathy was accompanied by impaired acetylcholine receptor clustering and reduced tyrosine kinase activity at motor nerve endings. A functional assay in myocytes derived from human fetuses confirmed that the variant blocks MUSK-dependent motor endplate formation. Taken together, the results strongly support a causal role of this founder mutation in MUSK, further expanding the gene set associated with FADS and offering new opportunities for prenatal genetic testing.

Neuromuscular disease. DOK7 gene therapy benefits mouse models of diseases characterized by defects in the neuromuscular junction.

The neuromuscular junction (NMJ) is the synapse between a motor neuron and skeletal muscle. Defects in NMJ transmission cause muscle weakness, termed myasthenia. The muscle protein Dok-7 is essential for activation of the receptor kinase MuSK, which governs NMJ formation, and DOK7 mutations underlie familial limb-girdle myasthenia (DOK7 myasthenia), a neuromuscular disease characterized by small NMJs. Here, we show in a mouse model of DOK7 myasthenia that therapeutic administration of an adeno-associated virus (AAV) vector encoding the human DOK7 gene resulted in an enlargement of NMJs and substantial increases in muscle strength and life span. When applied to model mice of another neuromuscular disorder, autosomal dominant Emery-Dreifuss muscular dystrophy, DOK7 gene therapy likewise resulted in enlargement of NMJs as well as positive effects on motor activity and life span. These results suggest that therapies aimed at enlarging the NMJ may be useful for a range of neuromuscular disorders.

DOK7 congenital myasthenic syndrome in childhood: Early diagnostic clues in 23 children

Mutations in DOK7 are a common cause of congenital myasthenia. Treatment with ephedrine or salbutamol is effective, but diagnosis is often delayed. The aim of our study was to find early clues to the diagnosis of DOK7 congenital myasthenic syndrome. We included 23 children of 20 families. Onset of symptoms ranged from birth to age 3years. 13 presented at birth with feeding difficulties, 11 with stridor (documented vocal cord palsy in 7), 3/11 with hypotonia/poor head control. Weakness was more pronounced proximally in all, axial in early presenting infants. Muscle biopsy showed non-specific features in 15/16, type 1 fibre predominance in 14/16, areas devoid of oxidative enzyme activity in 7/16. Muscle imaging was normal in 8/10, 2/10 showed mild non-specific changes. A diagnostic clue suggesting CMS rather than myopathy was the discrepancy between muscle imaging or histology findings compared with the degree of weakness. Repetitive nerve stimulation and stimulation single fibre electromyography were pathological in 9/17 and 13/14, respectively. In conclusion, stridor and feeding difficulties at birth or progressive weakness despite normal milestones in infancy point to the diagnosis and should lead to neurophysiological and genetic investigation. Fatigability can be absent or easily missed in the first years of life.

Salbutamol therapy in congenital myasthenic syndrome due to DOK7 mutation

IntroductionSalbutamol is a selective B2-adrenergic agonist, which has previously been described to be associated with partial improvement of myasthenia gravis and congenital myasthenic syndromes (CMS). In this study, we analyzed the effect of salbutamol in five patients with Dok-7 CMS. MethodsWe studied 5 patients (2 male and 3 female), with a mean age of 27±11.06years, who harbored c.1124_1127dupTGCC, p.G64R and/or p.S45L mutations in DOK7 gene. Salbutamol was given at a dose of 2mg three times daily (6mg/day) to all patients. The response was assessed by QMG score at baseline, 3, 6, 9 and 12months; ADL-MG score and 6minute walk test at baseline and after 12months during follow-up clinic visits. Side effect profile of salbutamol was also evaluated. ResultsWe noted an increasingly positive response as measured by the QMG score after 3months of salbutamol treatment. Improvement in specific subcomponents of the QMG score such as leg outstretched in 45° supine was most marked. In ADL-MG scores and 6minute walk test, comparison between baseline and after 12months revealed a clear beneficial response. Salbutamol was well tolerated in all patients. ConclusionsSalbutamol is an effective treatment in Dok-7 CMS. This study provides class IV evidence that salbutamol given at a dose 6mg/day improves function as measured by the QMG score, ADL-MG score and 6minute walk test.